A fundamental unsolved problem of cell biology and biochemistry is the molecular definition of the mechanisms of phospholipid transport for membrane biogenesis. This process is essential for all cell growth, replication, differentiation and homeostasis. The focus of this proposal is to use the power of yeast molecular genetics to dissect the process and understand the general principles of how it occurs throughout eukaryotic organisms. We have made progress in defining several of the genes and gene products that participate in the transport reactions, but large gaps in our knowledge still remain. The work will continue to concentrate on the transport reactions involving the aminoglycerophospholipids (phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine), since the tools we have previously developed greatly facilitate measuring inter-membrane transfer events, and also provide the basis of genetic screens and selections for strains that are defective in these transport processes. In the first Specific Aim we will further define the role of protein ubiquitination in regulating physical associations between the endoplasmic reticulum and the mitochondria, and in serving as a nucleation site for the assembly of multi-protein complexes involved in moving the lipids. In the second Specific Aim we will extend our findings from previous genetic, and protein-protein interaction studies, to reconstitute the associations between biological and synthetic donor membranes in vitro; and mechanistically define how individual proteins and lipids regulate the transport of specific aminoglcyerophospholipids. In the third Specific Aim we will continue to apply genetic and biochemical tools to elucidate the genes and gene products involved in the export of phosphatidylethanolamine from mitochondria and the Golgi apparatus. In the fourth Specific Aim we will use recently developed conditional alleles for phosphatidylserine decarboxylaseexpressed in mice, to define the function of this enzyme in mitochondrial biogenesis in mammalian systems. The combined genetic and biochemical approaches we propose will provide new mechanistic and molecular information about phospholipid transport processes in eukaryotic cells and provide new insights into the regulation of membrane biogenesis. This fundamental work is most relevant to areas of human disease concerned with control of cell growth and mitochondrial dysfunction, such as cancer and heart disease.